Xyloglucan is a major structural polysaccharide in the primary (growing) cell wall of plants. Structurally, xyloglucans consists of a cellulose-like beta-1,4-linked glucose backbone which is frequently substituted with various side chains. The xyloglucans of most dicotyledonous plants, some monocotyledons and gymnosperms are highly branched polysaccharides in which approx. 75% of the glucose residues in the backbone bear a glycosyl side chain at 0-6. The glycosyl residue that is directly attached to the branched glucose residue is invariably alpha-D-xylose. Up to 50% of the side chains in the xyloglucans contain more than one residue due to the presence of beta-D-galactose or alpha-L-fucose-(1-2)-beta-D-galactose moieties at 0-2 of the xylose residues (C. Ohsumi and T. Hayashi (1994) Plant and Cell Physiology 35:963-967; G. J. McDougall and S. C. Fry (1994) Journal of Plant Physiology 143:591-595; J. L. Acebes et al. (1993) Phytochemistry 33:1343-1345). On acid hydrolysis, the xyloglucan extracted from cotton fibers yielded glucose, xylose, galactose and fucose in the ratio of 50:29:12:7 (Hayashi et al., 1988).
Xyloglucans produced by solanaceous plants are unusual in that typical only 40% of the beta-1,4-linked glucose residues bear a glycosyl side chain at 0-6. Furthermore, up to 60% of the xylose residues are substituted at 0-2 with alfa-L-arabinose residues and some solanaceous plants, such as potato, also have xyloglucans with beta-D-galactose substituents at 0-2 of some of the xylose residues (York et al (1996)).
Xyloglucan is believed to function in the primary wall of plants by crosslinking cellulose-microfibrils, forming a cellulose-xyloglucan network. This network is considered necessary for the structural integrity of primary cell-walls (Carpita et al., 1993). Another important function of xyloglucan is to act as a repository for xyloglucan subunit oligosaccharides that are physiologically active regulators of plant cell growth. Xyloglucan subunits may also modulate the action of a xyloglucan endotransglycosylase (XET), a cell-wall associated enzyme that has been hypothesized to play a role in the elongation of plant cell walls. Therefore xyloglucan might play an important role in wall loosening and consequently cell expansion (Fry et al., 1992).
The seeds of many dicotyledonous species contain xyloglucan as the major polysaccharide storage reserve. This type of xyloglucan, which is localized in massive thickenings on the inside of the seed cotyledon cell wall, is composed mainly of glucose, xylose and galactose (Rose et al., 1996).
Seeds of the tamarind tree Tamarindus indica became a commercial source of gum in 1943 when the gum was found useful as a paper and textile size. Sizing of jute and cotton with tamarind xyloglucan has been extensively practiced in Asia owing to the low cost of the gum and to its excellent properties. Food applications of tamarind xyloglucan include use in confections, jams and jellies and as a stabilizer in ice cream and mayonnaise (Whistler et al., 1993).
Xyloglucanase activity is not included in the classification of enzymes provided by the Enzyme Nomenclature (1992). Hitherto, this enzymatic activity has simply been classified as glucanase activity and has often been believed to be identical to cellulolytic activity (EC 3.2.1.4), i.e. activity against .beta.-1,4-glycosidic linkages in cellulose or cellulose derivative substrates, or at least to be a side activity in enzymes having cellulolytic activity. However, a true xyloglucanase is a true xyloglucan specific enzyme capable of catalyzing the solubilisation of xyloglucan to xyloglucan oligosaccharides but which does not exhibit substantial cellulolytic activity, e.g. activity against the conventionally used cellulose-like substrates CMC (carboxymethylcellulose), HE cellulose and Avicel (microcrystalline cellulose). A xyloglucanase cleaves the beta-1,4-glycosidic linkages in the backbone of xyloglucan.
Xyloglucanase activity is described by Vincken et al. (1997) who characterizes three different endoglucanases from Trichoderma viride (similar to T. reesei) which all have high activity against cellulose or CMC and show that the EndoI (which is indeed belonging to family 5 of glycosyl hydrolases, see Henrissat, B. et al. (1991, 1993)) has essentially no (i.e. very little) activity against xyloglucan, and that EndoV (belonging to the family 7 of glycosyl hydrolases) and EndoIV (belonging to the family 12 of glycosyl hydrolases) both have activity against xyloglucan and CMC, respectively, of the same order of magnitude.
International patent publication WO 97/13862 describes two cellulases from Aspergillus niger strain N400 as described in EP-A 0 463 706. The sequence of lac12 can be ascribed to family 12, and the sequence of lac64 is determined as belonging to family 5 by comparison with known homologous cellulases in the EMBL data base. Both enzyme have cellulase and .beta.-glucanase activity. They have the highest activity against barley .beta.-glucan, and they both show good CMC activity and some xyloglucanase activity. Both enzyme has pH optima at 3.5 on CMC and 5.5 on barley .beta.-glucan.
International Patent Publication WO 94/14953 discloses a xyloglucanase (EG II) cloned from the fungus Aspergillus aculeatus and expressed in the fungus Aspergillus oryzae which has high xyloglucanase activity and very little cellulase activity. This EG II enzyme which shows xyloglucanase activity in the pH range 2.5-6 and optimum activity at pH 3-4 also belongs to family 12 of glycosyl hydrolases.
In summary, up till now xyloglucanase activity has only been found in fungal enzymes belonging to the families 7 and 12 of glycosyl hydrolases and exhibiting this activity in the acidic to near neutral pH range.
However, many important processes, either industrial or using industrially produced agents, are operating at an alkaline pH. Thus, it is an object of the present invention to provide a true xyloglucanase enzyme with a high xyloglucanase activity at an alkaline pH and essentially no activity on cellulose or cellulose derivatives.